LOCUS       sequence-235143-        6726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the tAKAR-gamma sensor for detecting PKA 
            activity using two-photon fluorescence lifetime imaging microscopy 
            (2pFLIM).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6726)
  AUTHORS   Ma L, Jongbloets BC, Xiong WH, Melander JB, Qin M, Lameyer TJ, 
            Harrison MF, Zemelman BV, Mao T, Zhong H
  TITLE     A Highly Sensitive A-Kinase Activity Reporter for Imaging 
            Neuromodulatory Events in Awake Mice.
  JOURNAL   Neuron. 2018 Aug 22;99(4):665-679.e5. doi: 
            10.1016/j.neuron.2018.07.020. Epub 2018 Aug 9.
  PUBMED    30100256
REFERENCE   2  (bases 1 to 6726)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.neuron.2018.07.020"; journalName: "Neuron"; date: 
            "2018-08-22- 22"; volume: "99"; issue: "4"; pages: "665-679.e5"
COMMENT     Sequence Label: pRK5_tAKAR-gamma
FEATURES             Location/Qualifiers
     source          1..6726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      943..952
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(949..951,952..954,955..1467)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     CDS             join(2371..2373,2374..2376,2377..2889)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNWNSVNVYIMADKQKNGIK
                     VNFKIRHNIE"
     regulatory      3026..3035
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3039..3173
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3076..3095)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3130..3149
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3242..3599
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3242..3433
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3265..3285)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3450..3585
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3619..3636)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3619..3635)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3628..3650)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3848..4303
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3935..3954)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4145..4166
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4320..4339)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4439..4461
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4499..4517)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4585..4689
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4690..4758,4759..5550)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4908..4927)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5721..6309
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6210..6229
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6463..6480
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6597..6618
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6633..6638,6639..6656,6657..6663)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6671..6687
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     6676..6698
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6695..6711
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6695..6711
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcccgggc tcgagaccgg tcgccaccat ggtgagcaag
      961 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1021 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1081 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1141 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1201 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1261 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1321 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1381 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     1441 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1501 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1561 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1621 gtgaccgccg cccgcatgca taagttttct caagaacaga tcggcgaaaa cattgtgtgc
     1681 agggtcattt gtaccacggg tcaaattccc atccgagatt tgtcagctga tatttcacaa
     1741 gtgcttaagg aaaaacgatc cataaagaaa gtttggacat ttggtagaaa cccagcctgt
     1801 gactatcatt taggaaacat ttcaagactg tcaaataagc atttccaaat actactagga
     1861 gaagacggta accttttatt gaatgacatt tccactaatg ggacctggtt aaatgggcaa
     1921 aaagtcgaga agaacagcaa tcagttactg tctcaaggtg atgaaataac cgttggtgta
     1981 ggcgtggaat cagatatttt atctctggtc attttcataa acgacaaatt taagcagtgc
     2041 ctcgagcaga acaaagttga tcgctctgca ggtaagccag gcagcggcga gggcagcacc
     2101 aagggcctgc gtcgcgccac cctggtggac ggcggcaccg gcggcagcga gctcatgggc
     2161 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2221 ctgcccgaca accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag
     2281 cgcgatcaca tggtcctgct ggagagagtg accgccgccg ggatcactct cggcatggac
     2341 gagctataca agggtggaac tggaggttca atggtgagca agggcgagga gctgttcacc
     2401 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2461 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagct catctgcacc
     2521 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta cggcctgatg
     2581 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     2641 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     2701 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2761 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactggaa cagcgtcaac
     2821 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     2881 aacatcgagg gtgaattcgg taaaaagaag aaaaagaagt caaagacaaa gtgtgtaatt
     2941 atgtaatcta ggggatccac cggaagccac ctgcatgcgg ccgcaattcc ccggggatcc
     3001 tctagagtcg acctgcagaa gcttggccgc catggcccaa cttgtttatt gcagcttata
     3061 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3121 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcgggaatt
     3181 aattcggcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct
     3241 tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     3301 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3361 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3421 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3481 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     3541 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     3601 agctgttaac agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     3661 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     3721 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct
     3781 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     3841 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     3901 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     3961 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4021 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     4081 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4141 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt
     4201 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     4261 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     4321 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     4381 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4441 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     4501 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     4561 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     4621 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     4681 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     4741 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     4801 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     4861 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     4921 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     4981 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5041 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5101 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5161 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5221 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5281 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5341 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     5401 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     5461 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     5521 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     5581 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     5641 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     5701 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5761 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     5821 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     5881 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     5941 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6001 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6061 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6121 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6181 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6241 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6301 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6361 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     6421 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     6481 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     6541 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     6601 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     6661 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga catgattacg
     6721 aattaa
//
